C-reactive protein deficiency ameliorates experimental abdominal aortic aneurysms

Background C-reactive protein (CRP) levels are elevated in patients with abdominal aortic aneurysms (AAA). However, it has not been investigated whether CRP contributes to AAA pathogenesis. Methods CRP deficient and wild type (WT) male mice were subjected to AAA induction via transient intra-aortic infusion of porcine pancreatic elastase. AAAs were monitored by in situ measurements of maximal infrarenal aortic external diameters immediately prior to and 14 days following elastase infusion. Key AAA pathologies were assessed by histochemical and immunohistochemical staining procedures. The influence of CRP deficiency on macrophage activation was evaluated in peritoneal macrophages in vitro. Results CRP protein levels were higher in aneurysmal than that in non-aneurysmal aortas. Aneurysmal aortic dilation was markedly suppressed in CRP deficient (aortic diameter: 1.08 ± 0.11 mm) as compared to WT (1.21 ± 0.08 mm) mice on day 14 after elastase infusion. More medial elastin was retained in CRP deficient than in WT elastase-infused mice. Macrophage accumulation was significantly less in aneurysmal aorta from CRP deficient than that from WT mice. Matrix metalloproteinase 2 expression was also attenuated in CRP deficient as compared to WT aneurysmal aortas. CRP deficiency had no recognizable influence on medial smooth muscle loss, lymphocyte accumulation, aneurysmal angiogenesis, and matrix metalloproteinase 9 expression. In in vitro assays, mRNA levels for tumor necrosis factor α and cyclooxygenase 2 were reduced in lipopolysaccharide activated peritoneal macrophages from CRP deficient as compared to wild type mice. Conclusion CRP deficiency suppressed experimental AAAs by attenuating aneurysmal elastin destruction, macrophage accumulation and matrix metalloproteinase 2 expression.


Introduction
Abdominal aortic aneurysms (AAA) are local dilation of aortic segments particularly infrarenal aorta resulted from media destruction and diagnosed when the diameter exceeds 50% of adjacent aortic diameter (1).AAAs progress asymptomatically but fatal upon premature rupture with an estimated annual death of 100,000 worldwide (1)(2)(3).While poorly defined, inflammation is crucial in AAA pathogenesis (1,4).
C-reactive protein (CRP) is an acute inflammatory protein and increases dramatically in response to almost tissue injury, infection and inflammation, thus being used as an inflammatory marker in clinic (5)(6)(7)(8)(9).CRP binds phosphorylated choline of pneumococcal C-polysaccharides, activates classical complement pathway or interacts with the Fc gammaR1/2 (FcgR1/2) receptor, altogether promoting the clearance of pathogens and cellular debris (10).CRP also modulates the function of immune cells including macrophages and lymphocytes (11)(12)(13)(14)(15). Thus, CRP is important for both host defense and human disease pathogenesis by regulating innate and adaptive immunity.
In clinical AAAs, it has been reported that the CRP levels were positively associated with aneurysm diameter (16)(17)(18)(19)(20). CRP was also highly expressed in aneurysmal as compared to nonaneurysmal aortas (21).Additionally, serum CRP levels have been used for helping AAA diagnosis as well as predicting clinical outcomes following AAA repair (22,23).However, it has not been investigated whether CRP mediates AAA pathogenesis.Therefore, this study assessed the influence of CRP deficiency on experimental AAA formation and progression in the intra-aortic elastase infusion-induced AAA model.

Materials and methods
Mice CRP deficient mice were previously generated using CRISPR/ Cas9 and homologous recombination technology to knock-in a STOP cassette at the ATG site of the CRP gene on C57BL/6 genetic background) at Shanghai Biomodel Organism Science & Technology Development Co., Ltd (Shanghai, China) (24).CRP deficient and C57BL/6 wild type (WT) mice were used for all experiments.The use and care of animals in this study were approved by the Laboratory Animal Management Committee of Xi'an Jiaotong University, Xi'an, China (No. 2022-623).

AAA creation in mice
Male CRP-deficient and WT control mice at 9 weeks of age were used for experiments.AAAs were induced in infrarenal aorta under sterile condition using porcine pancreatic elastase (PPE) infusion method as previously described (26)(27)(28).Briefly, mice were anesthetized by 2% isoflurane inhalation, and a laparotomy was created to expose the infrarenal aorta.Mice were infused with PPE Thereafter, aortotomy and laparotomy were sequentially closed using 10-0 and 6-0 silk sutures, respectively.Mice were recovered, housed in individual cages, and monitored daily for morbidity and mortality.

Measurements of aneurysmal aortic diameters
External infrarenal aortic diameters were measured in situ using a digital microscope.Briefly, infrarenal aortic segment was photographed immediately prior to (baseline) and 14 days following PPE.Maximal external infrarenal aortic diameters were determined using Motic Image Plus 3.0 ML software (Motic Electric Group Co., Ltd, Xiamen, China).An AAA was defined as a more than 50% increase in external diameter over the baseline level (29).

Statistical analysis
All statistical analyses were performed using the GraphPad software Version 9.0 (Boston, MA, USA).Data on continuous variables were presented as either mean ± standard deviation if normally distributed, and statistical significance was tested using Student's t-test, or one, two-way ANOVA followed by two group comparison tests.Otherwise, data were given as median and interquartile range, and statistical significance was determined using nonparametric Mann-Whitney test.Statistical significance level was set at p<0.05.

CRP protein is increased in experimental aneurysmal aortas
To examine whether CRP protein expression is altered in aneurysmal aortas, we stained aortic frozen sections from aneurysmal (PPE-infused) and non-aneurysmal WT mice.As depicted in Figure 1, rare or no positive staining was not seen in non-aneurysmal aorta.In contrast, an intense and diffusion CRP staining was noted in aneurysmal aortas (Figure 1).In serial sections, CRP staining was coincident with inflammatory cell accumulation in aneurysmal aortas.

CRP deficiency mitigates experimental aneurysmal aortic dilation
To clarify the effect of CRP in experimental AAAs, previously generated CRP-deficient (CRP -/-) mice were used in this study (24).The homozygotes of CRP-deficient mice were screened by PCR genotyping and Western blotting (Figure 2A).Luminal PPE infusion in infrarenal aorta was performed to induce AAAs in both WT and CRP -/-mice.Fourteen days following PPE infusion, aortic dilation, as measured by external aortic diameter, was seen in both WT and CRP -/-mice as compared to the baseline level.However, maximal external aortic diameter was significantly smaller in CRP -/-(1.08 ± 0.11 mm) than that in WT (1.21 ± 0.08 mm) mice on day 14 after PPE infusion (Figures 2B, C).When subtracting aortic dilation due to the pressed infusion (approximately 0.8 mm), CRP deficiency per se led to a 32% reduction in aneurysmal enlargement (Figure 2C).Even considering the influence of baseline aortic diameter, a remarkable reduction in external aortic diameter was observed in CRP -/-as compared to WT mice 14 days following PPE infusion (Figure 2D).Thus, CRP may in part mediate experimental aneurysmal expansion.

CRP deficiency has no recognizable impact on angiogenesis in aneurysmal aorta
Angiogenesis, an additional key AAA pathology, also contributes to the progression of AAAs (35,36).Angiogenesis, as determined by the density of CD31 + neovessels, was not differentiated between CRP -/- (50.82 ± 13.40/ACS) and WT (43.90 ± 12.23/ACS) mice (Figure 5).Thus, neoangiogenesis may have no or limited contribution to the suppression of experimental AAAs by CRP deficiency.

CRP deficiency limits classic macrophage activation in vitro
Classic macrophage activation (conventionally known as proinflammatory M1 macrophage activation/polarization) promotes AAA formation and progression (33,37).In classical activated macrophage by LPS, the expression levels of mRNA for TNF-a and COX-2, but not IL-1b, IL-6 (M1 marker genes) were significantly reduced in CRP deficient as compared to WT macrophages (Figure 6).However, no significant influences were found for the expression levels of all alternative activation macrophage markers (conventionally knowns as anti-inflammatory M2 macrophages) after activated by IL-4 (Supplementary Figure 1).

Discussion
Although the detailed mechanism of AAA pathogenesis is still unclear, the involvement of inflammation in the formation and progression of AAAs has become a consensus in this field (38).CRP, as one of the important inflammatory acute phase proteins, is involved in the development of many cardiovascular diseases (39)(40)(41)(42)(43)(44)(45).In this study, we found increased expression of CRP protein in experimental AAAs.CRP deficiency inhibited experimental AAA enlargement in the PPE infusion AAA model.Histologically, the suppression of experimental AAAs by CRP deficiency was associated with the attenuation of medial elastin destruction, aneurysmal wall macrophage accumulation and MMP2 expression.Additionally, CRP deficiency partially inhibited proinflammatory macrophage activation/polarization.Thus, our study indicated that CRP may in part mediate AAA pathogenesis.
In previous studies, CRP has been shown to regulate phenotypic differentiation and activity of macrophages (46)(47)(48).In patients with certain cardiovascular diseases, CRP expression levels was positively correlated with M1 macrophage activation (48).This was consistent with our findings that CRP deficiency downregulated the expression levels of M1 macrophages marker genes.Nuclear factor kappa B (NF-kB) regulates proinflammatory macrophages polarization as well as CRP activity, thus reduced M1 macrophage polarization due to CRP deficiency may potentially associate with altered NF-kB signaling activity (42,47,49,50).Additionally, reduced M1 macrophage activation in CRP deficient  CRP deficiency has no remarkable impact on angiogenesis in aneurysmal aortas.Frozen sections were prepared from the aortas of non-aneurysmal normal (upper left) and aneurysmal (wild type: upper right; CRP -/-: lower left) mice (n=10-11 mice per group) and stained with an anti-CD31 antibody to assess aneurysmal angiogenesis.Lower right panel: angiogenesis in wild and CRP -/-aneurysmal mouse aortas was quantitated as the number of CD31-positive neovessels per ACS.Data are mean ± standard deviation.Student's t tests, no significant difference between two groups.macrophages may attenuate the expression of proaneurysmal mediators including cytokines and MMPs consequently leading to experimental AAA inhibition.
We found that CRP expression was elevated in experimental AAA lesion.While we have no data showing the source for increased CRP, this may result from locally and/or systemically increased CRP as reported in other pathological condition (51, 52).Macrophages have been reported to be the main cellular source for non-liver derived CRP (53).CRP interacts with macrophage FcgR1/ 2 or lectin-like oxidized LDL receptor 1 (LOX-1) and bind to Oxidized low-density lipoprotein (Ox-LDL), thus regulating macrophage functional activity through multiple pathways and mediating vascular inflammatory diseases (10,(54)(55)(56)(57).Alternatively, the interaction of CRP with LOX-1 and Ox-LDL also enhances CRP expression in endothelial cells, which further promote vascular inflammation (58).Therefore, the potential modulation of macrophage activity by CRP may partially contribute to experimental AAA pathogenesis in the PPE AAA model.
Although the loss of endogenous CRP reduced experimental AAAs, the inhibition was not strong as demonstrated for other agents including metformin (59-61).Therefore, we need more experimental evidence to validate whether CRP can be a therapeutic target for AAA disease.In summary, this study demonstrates a partial role of CRP in experimental AAA pathogenesis of AAAs using CRP deficient mice.

2 CRP
FIGURE 2CRP deficiency suppresses experimental aneurysmal dilation.(A): Phenotype identification of CRP deficient mice.Phenotyping for CRP homozygotes (CRP deficient mice: CRP -/-), CRP heterozygotes (CRP +/-) and wild type (WT) mice using PCR assay.CRP expression levels in the livers of CRP -/-and WT mice were determined via qRT-PCR and Western blotting analyses.(B): Representative photographic images for infrarenal aortas of wild type and CRP -/-mice prior to and 14 days after the elastase infusion.AAAs were induced in male CRP -/-and its wild type control mice using intra-aortic infusion of PPE.Influence on AAAs were assessed via in situ measurements of maximal infrarenal aortic diameters.Dotted line indicates aortic expansion even after PPE solvent (PBS) pressed infusion (about 0.8 mm in our lab) (C, D): Maximal infrarenal aortic external diameters presented as absolute diameter on days 0 (baseline) and 14 after PPE infusion (C) or the percentage of diameters over baseline (D).n=10-11 mice per group.Two-ANOVA followed by two group comparison, **p<0.01compared to wild type mice at same timepoint (C).Student t-test, p=0.06 compared to wild type mice (D).(E): Representative aortic images for H&E (left panels), elastin via Elastic Van Gieson (middle panels), and SMCs via an anti-SMC a antibody immunostaining (right panels) from non-aneurysmal and aneurysmal (wild type and CRP -/-) mice.(F, G): Quantification of medial elastin degradation (F) and SMC depletion (G) scores (media and interquartile) of wild type and CRP -/-aneurysmal aortas.Nonparametric Mann-Whitney test, *p<0.05 compared to wild type mice.

TABLE 1
Primer sequences used for qRT-PCR assay.